KR20190058620A - Clearance of protein aggregates including phospholipase D and tau, and treatment of protein diseases - Google Patents

Abstract

This application discloses compounds that are active in self-sustaining flow and pharmaceutical compositions comprising such active agents. In addition, the use of such compounds and pharmaceutical compositions in the treatment of neurodegenerative diseases, particularly protein diseases and tauopathies, such as Alzheimer's disease, is disclosed. This further discloses a method for improving self-propelled flow.

Description

Clearance of protein aggregates including phospholipase D and tau, and treatment of protein diseases

This disclosure relates to compounds that are active in autophagic flux and to pharmaceutical compositions comprising such compounds. It further relates to the use of such compounds in the treatment of neurodegenerative diseases, particularly Alzheimer's disease.

Alzheimer's disease (AD) affects approximately 5 million Americans, and this number is expected to triple by 2050. Currently, there is no therapy for treating Alzheimer's or other related tauopathy. Clinical trials using immunotherapy targeting amyloid beta (Aβ) have had limited success, but only in the subset of ADs that have AD or other neurodegenerative diseases. In addition, there is no therapy to target other protein diseases, including other major neuropathological components of tau, AD. AD accounts for most of the dementia in individuals over 65 years of age, and it is estimated that hospice care for people with health care, long-term care, and AD and other dementia costs $ 226 billion annually. This vast economic and social burden does not account for the loss of the primary caregiver of a large number of families, including spouses and other family members.

Improvement in self-predisposition has therapeutic potential in the treatment of Alzheimer's disease. Autophagic flow (including the fusion of autosomal vesicles to lysosomes) is a novel modulator of autophagy because it causes reversal of clearance and pathophysiological reduction of protein aggregates. Thus, there is an ongoing need for a clearance of autologous digestive fluids with promoters and protein diseases of autophagic flow.

In some embodiments, the compounds disclosed herein, including pharmaceutically acceptable salts thereof, are provided.

In some embodiments, pharmaceutical compositions comprising a compound disclosed herein or a pharmaceutically acceptable salt thereof are provided. In another embodiment, the compounds and methods of preparing the pharmaceutical compositions are also provided, for example, in the examples provided below.

In some embodiments, there is provided a method of treating a neurodegenerative disease comprising administering to a subject in need thereof a therapeutically effective amount of a compound or pharmaceutical composition described herein.

In some embodiments, a method is provided for improving self-propelled flow. The method comprises the step of providing an effective amount of a compound or pharmaceutical composition described herein to a cell or protein aggregate.

These and other aspects of the invention are further disclosed in the following detailed description and examples.

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood with reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
Brief Description of the Drawings Figure 1 is a graph showing a photodiode array (PDA) spectrum of WHYKD8 in a mouse brain.
Figure 2 is a Western blot of the LC3-II level in primary cortical neurons after 6 hours of treatment with WHYKD1 (+/- BafA1) or WHYKD5.
Figure 3 depicts western blots of LC3-II, tau and p62 levels in organotypic slice cultures after 6 hours of treatment with WHYKD1 (upper) or WHYKD3, WHYKD5, WHYKD8, WHYKD9 or WHYKD12 drawing.
Figure 4 shows a bar graph showing the activation of phospholipase D (PLD) by the WHYKD series compound (10 [mu] M) and their ability to convert phospholipids to phosphatidyl ethanol in the presence of ethanol. C = control group, 12 = WHYKD12, 15 = WHYKD15, 19 = WHYKD19, 5 = WHYKD5, 8 = WHYKD8, Fipi = non-competitive inhibitor of PLD activity.
Figure 5 shows a bar graph showing the activation of phospholipase D (PLD) by the WHYKD series compound (1 [mu] M) and their ability to convert phospholipids to phosphatidyl ethanol in the presence of ethanol.

As a result of the intrinsic degradation process of macrophage predation, lipid changes that undergo self-encapsulating capsule dynamics are determined, although self-digestion mediates the engulfment and transfer of cytosolic components into lysosomes. We have previously shown that PLDl, which is primarily associated with the endosomal system, is partially renewed in the outer membrane of the autosomal vesicle-like structure with respect to nutritional starvation (Dall'Armi, 2010). The starvation of PLD1 as well as the starvation-induced increase of PLD activity is altered by bortmannin, a phosphatidylinositol 3-kinase inhibitor, suggesting that PLD1 may act downstream of Vps34. The pharmacological inhibition of PLD and genetic resection of PLD1 in mouse cells reduced the starvation-induced expansion of the LC3-positive compartment, consistent with the role of PLD1 in the regulation of autophagy. Furthermore, inhibition of PLD results in higher levels of tau and p62 aggregates in the trachea brain slice. These in vitro and in vivo findings establish a role for PLD1 in autoregulation.

In some embodiments, there is provided a compound having the formula (II), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

Wherein Y ¹ and Y ² are independently selected from the group consisting of CH and N;

X is selected from the group consisting of H, halides, and aryl;

R ¹ is selected from the group consisting of optionally substituted thioheteroaryl, hydroxyl-substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, The three carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N.

In some embodiments, the compound is selected from the group consisting of:

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In one embodiment the compound is as follows:

,

,

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In another embodiment, the compound is:

,

,

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In some embodiments, there is provided a compound having the formula (III), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

Wherein Y ¹ is CH;

Y ² is N;

X is a halide;

R ¹ is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 of said cycloalkyl Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N.

In some embodiments, the compound is selected from the group consisting of:

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In some embodiments, there is provided a compound having the formula (IV), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

X is a halide;

R ¹ is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 of said cycloalkyl Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N.

In some embodiments, the compound is selected from the group consisting of:

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In some embodiments, there is provided a compound having the formula (V), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

Wherein X is H;

R ¹ is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 of said cycloalkyl Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N.

In some embodiments, the compound is selected from the group consisting of:

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In some embodiments, there is provided a compound having the formula (VI), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof,

Wherein X is H;

R ¹ is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 of said cycloalkyl Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N.

In some embodiments, the compound is selected from the group consisting of:

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In some embodiments, there is provided a compound having the formula (VII), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

R ¹ is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 of said cycloalkyl Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N.

In some embodiments, the compound is selected from the group consisting of:

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In some embodiments, there is provided a compound having the formula (VIII), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

R ¹ is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 of said cycloalkyl Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N.

In some embodiments, the compound is selected from the group consisting of:

,

,

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In some embodiments, there is provided a compound having the formula (IX), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

Wherein Y ³ is CH or N;

R ² is optionally substituted (2-aminoethyl) aryl.

In some embodiments, the compound is selected from the group consisting of:

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In some embodiments, there is provided a compound having the formula (X), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

Wherein Y < ³ > is CH;

R ² is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N.

In some embodiments, the compound is selected from the group consisting of:

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In some embodiments, there is provided a compound having the formula (XI), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

R ² is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N.

In some embodiments, the compound is selected from the group consisting of:

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In some embodiments, there is provided a compound having the formula (XII), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

Y < ⁴ > is CH or N;

R ³ is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N.

In some embodiments, the compound is selected from the group consisting of:

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In some embodiments, there is provided a compound having the formula (XIII), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

R ² is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N.

In some embodiments, the compound is selected from the group consisting of:

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In some embodiments, there is provided a compound having the formula (XIV), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

R ² is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N.

In some embodiments, the compound is selected from the group consisting of:

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In some embodiments, there is provided a compound having the formula (XV): < EMI ID =

Wherein X is H or a halide;

Z ¹ is O;

로 이루어진 군으로부터 선택된다.R ⁴ is H, optionally substituted alkyl, Et, CF ₃ , optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and

≪ / RTI >

In some embodiments, the compound is selected from the group consisting of:

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In one embodiment the compound is as follows:

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof.

In some embodiments, pharmaceutical compositions comprising a compound disclosed herein or a pharmaceutically acceptable salt thereof are provided.

In some embodiments, there is provided a method of treating a neurodegenerative disease comprising administering to a subject in need thereof a therapeutically effective amount of a compound or pharmaceutical composition described herein. In some embodiments, the neurodegenerative disease is a protein disorder. Protein disorders include Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Huntington's disease, chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), inclusion body myocardial disease (IBM) Including but not limited to: amyloid angiopathy, prion disease, familial dementia, CADASIL, amyloidosis, Alexander's disease, seipinopathy, type II diabetes, alveolar proteinosis, cataract, cystic fibrosis and sickle- , But are not limited to these. In some aspects of this embodiment, the protein disease is tauopathy. Tauopathy is characterized by a variety of conditions including Alzheimer's disease, Parkinson's disease, Huntington's disease, progressive supranuclear palsy, chronic traumatic encephalopathy (CTE), frontotemporal dementia (FTD), Lytico-Bodig disease, subacute sclerosing panencephalitis, , Ganglioneuomas, and silver-sensitized particle disease. In a preferred embodiment, the neurodegenerative disease is Alzheimer ' s disease.

In some embodiments, a method is provided for improving self-propelled flow. The method comprises the step of providing an effective amount of a compound or pharmaceutical composition described herein to a cell or protein aggregate.

The embodiments described in this disclosure can be combined in various ways. Any aspect or feature described with respect to one embodiment may be incorporated into any other embodiment described in this disclosure. While various novel features of the principles of the invention have been set forth and described and pointed out as applied to the specific embodiments thereof, it is to be understood that various omissions and substitutions and changes may be made by those skilled in the art without departing from the spirit of the disclosure Should be understood. Those skilled in the art will recognize that the principles of the invention may be practiced otherwise than as described for the purpose of illustration and without limitation.

Example

The following examples are provided to further illustrate certain aspects of the present invention. These embodiments are illustrative only and are not intended to limit the scope of the invention in any way.

Example 1

Example Synthesis Plans

Scheme 1 illustrates a compound of the formula:

For example, the synthesis of compounds of formula (II) and (III).

Scheme 1

Typical general procedure

4-Chloroquinazoline and thiol were stirred at room temperature in dry THF. A base such as triethylamine was added. The reaction mixture was heated to 80 < 0 > C and then stirred at this temperature overnight, after which it was cooled to room temperature. Subsequently, after diluting with distilled water, the organic material was extracted with ethyl acetate (3x). The combined organic extracts were washed with brine (1x) and then dried over anhydrous sodium sulfate. The solvent was evaporated in vacuo and the crude material was purified by column chromatography or preparative TLC using 10% MeOH in methylene chloride or 10: 1 pentane: diethyl ether as eluent.

The above procedure is shown. Other embodiments disclosed herein could be made by similar techniques or by other methods known in the art.

 Scheme 2 shows the preparation of 1-chloro-7-fluoroisoquinoline.

Scheme 2

Scheme 3 illustrates a compound of the formula:

For example, the synthesis of compounds of formula (IV), (V), (VI), (VII) and (VIII).

Scheme 3

Scheme 4 illustrates a compound of the formula:

For example, the synthesis of compounds of formula (XII) and (XIII).

Scheme 4

Scheme 5 depicts a compound of the formula:

,

,

For example, the synthesis of compounds of formula (IX), (X) and (XI).

Scheme 5

Scheme 6 illustrates a compound of the formula:

,

,

For example, the synthesis of a compound of formula (XIV).

Scheme 6

Example 2

Self-predation  The active form of the flow and the phospholipase D

The WHYKD series of compounds were synthesized for optimal brain penetration based on molecular weight (MW) and partition coefficient (log P) according to Lipinski's Rule for CNS penetration: MW 400, log P 5.

An activator according to the following formula was synthesized according to the above scheme:

The molecular weight and log P were calculated. The results are shown in Table 1 below.

An activator according to the following formula was synthesized according to the above reaction formula.

 The molecular weight and log P were calculated. The results are shown in Table 2 below.

An activator according to the following formula was synthesized according to the above reaction formula.

The molecular weight and log P were calculated. The results are shown in Table 3 below.

An activator according to the following formula was synthesized according to the above reaction formula.

The molecular weight and log P were calculated. The results are shown in Table 4 below.

Example 3

Design of derivatives

Several series of derivatives were synthesized based on the following lead compounds:

In addition to log P, the topological polar surface area (tPSA), CLogP (log P calculated by the group contribution method) and LogS (solubility) were calculated. The results are shown in the following table.

Example 4

Biological testing of WHYKD compounds

The following enumerated assays were carried out using transfected HEK 293 (human embryonic kidney) cell lines engineered to express fluorescently tagged (mKate2) tau (not otherwise indicated). Cells were grown in the presence of the antibiotic doxycycline. When antibiotics are removed, the cells produce tau (which can be quantified) and thus allow comparison of the effect of the test compound on tau production. To facilitate adequate tau production, the doxycycline was removed for 72 hours prior to exposure to the test compound. Subsequently, the cells were plated on plates for each assay described below.

Self-feeding, aggregates and tau-mKate2 IC50 assay

Plated Tet - Adjusted HEK  Preparation of 293 cells (Jump-In (TM) cells)

1) HEK cells with tet-regulated expression of mKate2 tagged tau were grown in DMEM medium (Dulbecco's modified Eagle's medium) containing 0.5 μg / ml doxycycline (Dox) (MP-Bio # 198955) After 5 days, Dox was removed by washing the cells twice with sterile phosphate buffered saline (PBS; Invitrogen # 14190-144) and the cells remained in DMEM without Dox for 72 hours To further cluster the remaining Dox).

2) Coating 96-well, black plates (Costar # 3720) with ultra-thin clean bottoms with poly D lysine (PDL) (Sigma - p0899-M wt. 70,000-150,000) Lt; RTI ID = 0.0 > polystyrene / glass < / RTI > After 2 hours of inhalation of the PDL, the plate was again dried for 2 to 3 hours. The coating procedure was completed under sterile conditions. Plates were used immediately after coating.

3) Cells were desorbed from the flask using triple express (Invitrogen # 12605-010) and plated in PDL-coated 96 well plates. For plating, 200 [mu] l of medium was added to each well at a concentration of 400k cells / ml (80k cells / well). One row of peripheral wells was devoted to all aspects to prevent any changes in experimental conditions due to evaporation from these wells. Warm medium / PBS (without cells) was pipetted into the surrounding wells and then PBS was pipetted into the space between the wells to maintain homogeneous conditions throughout the center wells. The cells were then allowed to settle and adhered to the bottom of the plate for 18-24 hours.

Treatment with Cyto-ID (trademark) and dyeing

The Cyto-ID (brand name) assay measures autophagy and monitors autophagic flow in living cells inhibited by lysosomes using a novel dye that selectively labels the accumulated autologous cells. The dye used in the kit prevents its accumulation in the lysosome, but it allows the labeling of vacuoles associated with the self-trapping pathway using LC3 biomarkers.

The test compounds were added to the cells (with CytoID < (R) > dye and Hoechst staining) and then incubated for 3 hours. The reference compound was also used on each plate, and rapamycin was used for autophagic induction and then chloroquine was used for lysosomal inhibition. After 3 hours, the test compound was inhaled and then maintained. Cells were then washed and then read using a fluorescent plate reader. Chest, Cyto-ID (TM) and mKate2 were measured on a plate reader using three distinct wavelengths. The Hobest measurement allowed the normalization of the results across the wells. After reading, the test compound was added again and then the cells were left for an additional 21 hours. At the 24 hour time point cells were washed once more and then read using a fluorescent plate reader.

1) Prior to commencing treatment, warm FluoroBrite with 10% fetal bovine serum, dialyzed (FBS; Invitrogen # 26400-044) and 1% MEM (minimal essential medium) NEAA (nonessential amino acid) All wells were washed once using a (trademark) (FB) DMEM (Dulbecco's modified Eagle's medium) (Invitrogen # A18967-01). 280 [mu] l warm medium / PBS was maintained in the surrounding wells.

2) Test samples were prepared in warm Fluoroblot (TM) DMEM and in 10% FBS and 1% NEAA (FB-DMEM). Rapamycin 200 nM (Enzo # BML-A275-0025) was used to induce autophagy and 15 mM chloroquine was used for lysosomal inhibition (without the use of barfylomycin or similar compounds, they provided false negative results in the assay Because). Dimethylsulfoxide 1: 1000 (DMSO; Fisher # BP231-100) was used as a control. Self-digesting bombarder Cyto ID ™ (1: 500; Enzo # ENZ-51031-k200) and chest (1: 200) were added to these test sample preparations prior to cell treatment.

3) The treatment groups were staggered to obtain similar conditions across all groups. For n = 6, the three control wells were nearly the periphery of the plate, three were about the center of the plate, and were the same for rapamycin and any other drug treatment.

4) Cells were treated / stained at 37 ° C for 3 hours. At 3 hours after treatment, the test sample formulation containing Cyto-ID (TM) was carefully aspirated and transferred to fresh sterile microplates (Falcon # 353072) for reuse. The medium in the treatment plate was immediately replaced with warm FB-DMEM. A new plate with the test compound preparation was placed in an incubator at 37 占 폚.

5) The treatment plate was then rapidly washed three times with warm FB-DMEM (100 [mu] l / well). After the third wash, 50 占 퐇 of warm medium was left in each well. At this point, the plates were ready for reading. Please refer to the detailed description of the reading plate section. The quartz was read at excitation wavelength (Ex) = 355 nm and emission wavelength (Em) = 446 nm. mKate2 was read at Ex = 575 nm and Em = 630 nm. Cyto-ID (trade name) was read at Ex = 463 nm and Em = 534 nm.

6) Subsequently, the medium was replaced with the corresponding wells of the plate containing the test compound / dye preparation and then reincubated at 37 ° C.

7) After inhalation of all media 24 hours after the reaction, the plates were washed 3 times with warm FB-DMEM and incubated in 80 μl of FB-DMEM with either Hoechst (Ex = 355 nm Em = 446 nm), CytoID (Ex = 463 nm; Em = 534 nm) and mKate2 (Ex = 575 nm; Em = 630 nm).

Proteostat (TM) Protein Coagulation Assay

Aggresome was detected by measurement of p62 using proteostat (trademark) analysis. Agritis is an inclusion body formed when the ubiquitin-proteasome mechanism is covered by a protein that tends to aggregate. Typically, aglysomes are formed in response to some cellular stress, such as exposure to a solid temperature, viral infection, or reactive oxygen species. Agrillos can provide cytoprotection by sequestering toxic, aggregated proteins and can also facilitate their ultimate removal from cells by autophoresis. After reading the final plate described in the Cyto-ID (trademark) analysis described above, proteostat (TM) detection reagent was added to all wells and the plate was incubated for 15 minutes. After this incubation, the plate was read by the fluorescent plate reader at the specified wavelength. After this plate reading, the cells were fixed by incubation with warm paraformaldehyde for 8 minutes. The fixed cells were then read by a plate reader as before.

8) A detection solution was prepared by adding 10 μl Proteostat ™ Detection Reagent (ENZ-51023-KP002) and 200 μl of 10 × Assay Buffer to 1790 μl water and mixing the wells.

9) was added per well (each well has 80 [mu] l of Fluorobrite (TM) medium without FBS) and incubated in the dark for 15 minutes at room temperature.

10) Fluorescence (Ex = 550 nm; Em = 600 nm) was read for the proteostat (trade name).

11) Plates were fixed by adding 100 [mu] l of warm 4% paraformaldehyde (PFA; EMS # 15710) to all wells and incubated at room temperature for 8 minutes. After removing the PFA, the plate was washed three times with PBS (room temperature).

12) Plates were read back into fixed cells and fixed steric structures in a plate reader.

Note: CytoID (trademark) and proteostat (trade name) were measured for LC3 and p62, which can be replaced with corresponding fluorophore using fluorescently tagged antibodies.

Reading plate using Tecan M200

13) The plates were transferred to a plate reader (Tecan Infinite M200) and then read with optimal gain for mKate2, Cyto ID ™ and Proteostat ™ (last read only).

14) For each of the three signals, each well was read at five consistent locations per well. Each of these 5 locations lit 25 times. The signal from each well was first recorded as the average of 25 flashes, and the final value was based on an average of five readings per well. A perimeter of 1000 mu m was devoted to all wells to move any discrepancies in readings due to ancillary cell loss across the periphery resulting from inhalation and washing. Peripheral wells were used to detect any background noise due to the medium or PBS.

15) mKate2 was read at Ex = 575 nm and Em = 630 nm. Cyto-ID (trade name) was read at Ex = 463 nm and Em = 534 nm. For final reading, proteostat (trade name) was read at Ex = 490 nm and Em = 600 nm. Excitation and emission for all three readings were 9 nm and 20 nm, respectively.

16) The calculation was initially subtracted from the well background (medium alone well) and normalized using the Hobest level.

Plate reading using IN Cell Analyzer 2000 (high content imaging).

17) plates were transferred to in-cell analyzer 2000 and then imaged for mKate2, Cyto ID ™, Proteostat ™ and Hoechst (final reading only).

18) Each well was imaged in four consistent fields located around the center of the well. All images were taken using a 20x objective lens. The average readings from these four chapters were recorded as readings for the corresponding wells. No images were taken from the periphery of the well to move any discrepancies in readings due to ancillary cell loss across the periphery resulting from inhalation and washing. Peripheral wells were used to detect any background noise due to the medium or PBS.

19) Cyto ID (trademark) was imaged using a FITC / FITC filter (Ex = 490 nm - bandwidth 20 nm / Em = 525 nm - bandwidth 36 nm). MKate2 was imaged using a TexasRed / TexasRed filter (Ex = 579 nm - bandwidth 34 nm / Em = 624 nm - bandwidth 40 nm). The FITC / dsRed combination was used for the Proteostat (trade name) image (Ex = 490 nm - bandwidth 20 nm / Em = 605 nm - bandwidth 52 nm). Nuclei were imaged using a DAPI / DAPI filter set (Ex = 350 nm - bandwidth 50 nm / Em = 455 nm - bandwidth 50 nm).

p62 agglutinates and tau agglomerates western blot analysis

Western blot analysis was performed to determine protein changes in tau and p62. The cells were incubated as above and then incubated with the test compound for 24 hours. Test Compounds After incubation, the test compounds were inhaled and then the cells were washed before harvesting. The cells were agitated at low speed and then the supernatant was aspirated to leave the cell pellet. The cell pellet was then homogenized in buffer, then centrifuged at high speed, and the supernatant was inhaled and then further separated into a total fraction and an aggregate fraction to permit quantification of soluble and insoluble proteins. Western blotting was performed on the sample, then the gel was transferred to nitrocellulose and incubated with antibodies against Tau and p62. After incubation with the secondary antibody for detection, protein bands were quantified by chemiluminescence.

One) Jump-in (trade name) cells (see above) were maintained in 0.5 μg / ml doxycycline until use.

2) Three days before plating, cells were replated in a 40% confluence in the medium without the doxycycline.

3) On the day before the experiment, 750 000 cells were plated per well in 6-well plates (250 000 cells in 12 well plates).

4) On the day of the experiment, the cells were washed twice in warmed HBSS (Hank's Balanced Salt Solution) and then the medium containing the test compound (n = 3 / compound / 12 < / RTI > well). Plates were incubated with 5% CO ₂ at 37 ° C for 24 hours.

5) Cells were rinsed twice in warmed HBSS, then harvested in 1 ml HBSS and transferred to a 1.5 ml micro tube.

6) Samples were stirred at 500 xg for 2 min at 4 [deg.] C, and HBSS supernatant was inhaled to leave only the cell pellet. Cell pellets can be frozen instantly and stored at -80 ° C until use.

Sample preparation

7) Cell pellets were homogenized in a RIPA + (radio-immunoprecipitation assay) buffer containing protease inhibitor and phosphatase inhibitor and then gently homogenized using a cell homogenizer.

8) The sample was then centrifuged at 20,000 g for 20 minutes at 4 占 폚.

9) The supernatant was transferred to a new tube.

10) The supernatant was quantified for protein concentration using Pierce (TM) protein assay.

11) Total fraction : 20 μl of 1M DTT (dithiothreitol) (to make the final concentration of 100 mM DTT) and 50 μl of 4 × Invitrogen (pH 7.5) by adding RIPA + buffer to a volume of 130 μl and with 100 mM DTT 200 μg of supernatant was used to prepare a 1 mg / ml protein solution by adding NuPAGE LDS (lithium dodecyl sulfate) loading buffer (4X, 10 ml - NP0007).

12) Agglomerate fraction : For the agglutinates, a sample of 100 μg resulted in a final volume of 900 μl.

13) 100 [mu] l of 10% sarcosine solution was added to the sample and then stirred at 4 [deg.] C for 60 minutes.

14) The sample was then centrifuged at 100,000 g for 60 minutes at 4 < 0 > C.

15) The supernatant was carefully removed to leave unaffected pellets. The tube was turned over to remove any additional liquid. If there is an excess of liquid, steps 12-14 are repeated to ensure a pure coagulum sample. The pellet was resuspended and then solubilized in 65 μl of PBS followed by addition of 10 μl of 1 M and 25 μl of 4 × Invitrogen NuPAGE LDS loading buffer with 100 mM DTT.

Electrophoresis / Western blot

16) Before loading, the sample was heated at 90 DEG C for 2 minutes. Rapid agitation of the sample was performed to ensure no condensation on the tube. 4-12% Tris-Bis gel was prepared using 1X MOPS buffer (Invitrogen-NP0001) and antioxidant (NP0005). For the soluble fraction, 2 [mu] g samples were loaded into each well and 5 [mu] g samples were loaded per well for the insoluble fractions. Subsequently, 8 μl of the InvitrogenSharp MW standard was loaded (optimally all wells had the same volume of sample buffer).

17) The sample was electrophoresed at 150V for approximately 1 hour and 15 minutes (until the running dye reached the base of the gel)

18) The gel was then equilibrated for 5 minutes in transfer buffer (25 mM Tris-HCl pH 8.3, 192 mM glycine, 20% (v: v) methanol).

19) The gel was then removed and then transferred onto 0.2 μM nitrocellulose (GE BA83 10600001) at 200 mA for 90 minutes.

20) After delivery, blots were simply stained with 0.1% Ponceau S in 5% acetic acid to ensure a consistent sample loading between (15s) lanes.

21) The blots were then rinsed in TBS-T (Tris-buffered saline-polysorbate) for 2 minutes to remove the Fontos' stain before immunoblotting.

Immunodetection

22) Samples were blocked in 5% milk in TBST for 30 minutes and then rinsed in TBS-T until all buffer was clear (milk residue remained in solution).

23) The blots were incubated on the rocking table at 4 ° C with primary antibodies (1: 4000 PHF1 or CP27 for tau, 1: 4000 p62 Abnova for p62) in SuperBlock ™ TBS- ; And 1: 5000 GAPDH (glyceraldehyde 3-phosphate dehydrogenase) for the load control).

24) After primary antibody exposure, blots were washed three times for 15 min in TBS-T.

25) The blots were then incubated in a 1: 4000 secondary antibody (Jackson Laboratories goat anti-mouse HRP conjugate).

26) After the second round, the blot was washed three times for 15 minutes in TBST.

27) The blots were then developed using a Millipore chemiluminescent fluid (1 ml per reagent; WBKLS0500) and detected using Fuji LAS3000 imaging units in 10 second increments.

28) Images were quantified using retention software or NIH Image J.

PLD analysis

Cells were incubated with test compounds for 24 hours. At 25 minutes prior to collection at 24 hours, a 3% ethanol solution was added to the wells to catalyze the cleavage of the phospholipids. The wells were then placed on ice and harvested after washing. The collected wells were centrifuged, then the supernatant was aspirated and the pellet was retained. The pellet was resuspended and then chloroform / methanol lipid extraction was performed. After centrifuging the sample, the organic layer was separated, dried under nitrogen and stored at -80 占 폚. On the day of analysis, the samples were resuspended and analyzed by LC / MS. All phosphatidylethanol species (PEtOH 32-40: 0-6: 16/18: 0/1) were combined together and then expressed as total lipid content (all lipid species).

Analysis was performed on e18 primary cortical neurons from PLDl or PLD2 KO mice cultured for 14 days. Alternatively, an assay may be performed on the cells described above in excess PLD1 or PLD2 inhibitors, making it impossible for a particular action to contribute to the drug effect.

One) Jump-in (trade name) cells (see above) were maintained in 0.5 μg / ml doxycycline until use. For neurons, e18 embryos were used and cortical neurons were extracted and placed on PLD-collagen coated 6 well plates and incubated for 14 days prior to use.

2) Three days before plating, cells were replated in a 40% confluence in the medium without the doxycycline.

3) On the day before the experiment, 750 000 cells were plated per well in 6-well plates (250,000 cells in 12 well plates).

4) On the day of the experiment, the cells were washed twice in warmed HBSS and the medium containing the test compound (n = 3 / compound / dose), inhibitor (355 nM ML298 or 50 nM VU0150669) , 600 [mu] L in 12 wells). Plates were incubated with 5% CO ₂ at 37 ° C for 24 hours.

5) Twenty-five minutes before collection, 165 [mu] l of 3% ethanol solution was added to each well.

6) For collection, the plate was placed on ice and the cells were rinsed twice with ice-cold HBSS and then taken in 1 ml of HBSS and transferred to a 1.5 ml micro tube.

7) Samples were stirred at 500 xg for 2 min at 4 [deg.] C, and HBSS supernatant was inhaled to leave only the cell pellet. Cell pellets were frozen for an instant and stored at -80 ° C until use.

Example 5

Detection and Results of WHYKD Compounds

A photodiode array (PDA) was used to detect WHYKD8 in the mouse brain (Fig. 1). Samples were easily detected at different times on a peak (left) and under measurable curve area (AUC).

LC3-II levels were measured in primary cortical neurons after 6 hours of treatment with WHYKD1, WHYKD5, or WHYKD1 + BafA1 (Fig. 2). The presence of LC3-II is an indication of self-predation.

LC3-II levels were then measured in organotypic slice cultures 6 hours after WHYKD1 treatment (Fig. 3, top panel). Other compounds of the WHYKD series produced similar results (Figure 3, bottom panel). The RFP is a tag on the tau protein and can also be probed.

These experiments indicate that the WHYKD series of compounds induces self-predation and can reduce its aggregate substitute p62 as well as the aggregated form of tau.

PLD activation converts phospholipids to phosphatidyl ethanol in the presence of ethanol. This conversion was measured to show that the WHYKD series of compounds activates PLD at 10 μM concentration (FIG. 4) and 1 μM (FIG. 5). FIPI was a noncompetitive inhibitor of PLD activity and was used as a negative control.

All patents, patent applications and publications cited above are incorporated herein by reference in their entirety as if fully recited herein.

The present invention is thus described, and it will be clear that the same method can be changed in other ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention, and all such modifications are intended to be included within the scope of the following claims.

Claims (37)

A compound having the formula (II), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

Wherein Y ¹ and Y ² are independently selected from the group consisting of CH and N;
X is selected from the group consisting of H, halides, and aryl;
R ¹ is selected from the group consisting of optionally substituted thioheteroaryl, hydroxyl-substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, The three carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N. The compound of claim 1, wherein said compound is selected from the group consisting of: a salt, an enantiomer, a racemate, a mixture, or a combination thereof;

The compound according to claim 1, wherein the compound is

A compound, or a salt, an enantiomer, a racemate, a mixture, or a combination thereof. The compound according to claim 1, wherein the compound is

A compound, or a salt, an enantiomer, a racemate, a mixture, or a combination thereof. A compound having the formula (III), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

Wherein Y ¹ is CH;
Y ² is N;
X is a halide;
R ¹ is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 of said cycloalkyl Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N. 6. A compound according to claim 5, wherein the compound is selected from the group consisting of: a salt, an enantiomer, a racemate, a mixture,

A compound having the formula (IV), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

X is a halide;
R ¹ is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 of said cycloalkyl Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N. 8. A compound according to claim 7, wherein said compound is selected from the group consisting of: EMI58.1 or a salt, enantiomer, racemate, mixture,

A compound having the formula (V), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

Wherein X is H;
R ¹ is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 of said cycloalkyl Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N. 10. A compound according to claim 9, wherein the compound is selected from the group consisting of: a salt, an enantiomer, a racemate, a mixture,

A compound having the formula (VI), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

Wherein X is H;
R ¹ is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 of said cycloalkyl Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N. 12. The compound of claim 11, wherein the compound is selected from the group consisting of: a salt, an enantiomer, a racemate, a mixture,

A compound having the formula (VII), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

R ¹ is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 of said cycloalkyl Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N. 14. A compound according to claim 13, wherein the compound is selected from the group consisting of: a salt, an enantiomer, a racemate, a mixture,

A compound having the formula (VIII), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

R ¹ is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 of said cycloalkyl Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N. 16. The compound of claim 15, wherein the compound is selected from the group consisting of: a salt, an enantiomer, a racemate, a mixture,

A compound having the formula (IX), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

Wherein Y ³ is CH or N;
R ² is optionally substituted (2-aminoethyl) aryl. 18. The compound of claim 17, wherein the compound is selected from the group consisting of: a salt, an enantiomer, a racemate, a mixture,

A compound having the formula (X), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

Wherein Y < ³ > is CH;
R ² is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N. 20. The compound according to claim 19, wherein said compound is selected from the group consisting of: a salt, an enantiomer, a racemate, a mixture,

A compound having the formula (XI), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

R ² is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N. 22. A compound according to claim 21, wherein said compound is selected from the group consisting of: a salt, an enantiomer, a racemate, a mixture,

A compound having the formula (XII), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

Y < ⁴ > is CH or N;
R ³ is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N. 24. The compound of claim 23, wherein said compound is selected from the group consisting of: a salt, an enantiomer, a racemate, a mixture, or a combination thereof;

A compound having the formula (XIII), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

R ² is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N. 26. The compound of claim 25, wherein the compound is selected from the group consisting of: a salt, an enantiomer, a racemate, a mixture,

A compound having the formula (XIV), or a salt, enantiomer, racemate, mixture thereof, or a combination thereof:

R ² is selected from the group consisting of optionally substituted thioheteroaryl, optionally substituted (2-aminoethyl) aryl, halide, optionally substituted thiocycloalkyl and thioaryl, wherein 1 to 3 Carbon atoms are optionally substituted with a heteroatom selected from the group consisting of O, S, and N. 28. The compound of claim 27, wherein the compound is selected from the group consisting of: a salt, an enantiomer, a racemate, a mixture,

A compound having the formula (XV):

Wherein X is H or a halide;
Z ¹ is O;
R ⁴ is H, optionally substituted alkyl, Et, CF ₃ , optionally substituted cycloalkyl, optionally substituted aryl, optionally substituted heteroaryl, and

≪ / RTI > 30. The compound of claim 29, wherein said compound is selected from the group consisting of:

Or a salt, an enantiomer, a racemate, a mixture, or a combination thereof. 30. The compound of claim 29, wherein said compound is

A compound, or a salt, an enantiomer, a racemate, a mixture, or a combination thereof. 31. A pharmaceutical composition comprising a compound of any one of claims 1 to 31 or a pharmaceutically acceptable salt thereof. Comprising administering an effective amount of a compound of any one of claims 1 to 31 or a pharmaceutical composition of claim 32 to a subject in need of treatment for a neurodegenerative disease. 34. The method of claim 33, wherein said neurodegenerative disease is a protein disease. 35. The method according to claim 34, wherein the protein disease is tauopathy. 34. The method of claim 33, wherein said neurodegenerative disease is Alzheimer's disease. Providing a cell or protein aggregate with an effective amount of a compound of any one of claims 1 to 31 or a pharmaceutical composition of claim 32. A method of enhancing autophagic flux comprising:

Images